This proposal is aimed at developing two gene-replacement mouse models ? one that expresses a wildtype human Noxa protein and a second that expresses an apoptosis-deficient version of human Noxa, by replacing the murine NOXA gene and promoter sequence with the corresponding region of human NOXA using a bacterial artificial chromosome (BAC). Human (h) Noxa, a 54-residue protein, was initially identified as a ?pro-apoptotic? member of the Bcl-2 family, that could interact with pro-survival family member Mcl-1 via its BH3 domain to promote apoptosis. However, it was discovered later that hNoxa also promotes growth and proliferation in human hematopoietic lineage cells. This growth-promoting function, evident in both normal and malignant hematopoietic cells, has been attributed to Noxa?s ability to reprogram metabolism. In T cell and other leukemias, hNoxa is phosphorylated on a single serine (S13) by a glucose-dependent kinase. This eliminates its ability to bind Mcl-1 and promote apoptosis but enhances its ability to support metabolic reprogramming that increases glucose and glutamine uptake for growth and proliferation. Recent data point to a similar growth-promoting metabolic role for Noxa in primary human T cells following antigenic stimulation, leading to their rapid expansion and differentiation. Taken together, our studies point to a central and unique role for hNoxa in T cell development, expansion and differentiation. Its dual opposing functions make hNoxa a potential target for cancer therapy and for immunotherapy. Mouse models will be essential for in vivo validation and for further defining Noxa?s role in apoptosis and metabolism. Mouse (m)Noxa, differs from hNoxa, both structurally and in its regulation, has not been shown to promote growth or reprogram metabolism. In the first aim, we will generate hNOXA gene-replacement (GR) mice by replacing the entire mouse NOXA gene and promoter region with the corresponding region of hNOXA. An ES cell line will first be generated in which a portion of the mouse genome harboring the NOXA gene is deleted and replaced by a site-specific recombination target cassette. Next, the syntenic NOXA genomic sequence from the human genome will be inserted into the cassette in the prepared ES cell line. The second aim will be directed at determining whether hNoxa is accurately expressed and functional in the WT hNOXA mouse. Initial testing will focus on Noxa expression in CD8 T lymphocytes following in vitro activation, and on the impact of hNoxa expression on the generation, function and maintenance of effector and memory CD8+T cells in vivo. The DA hNOXA GR mouse, expressing an apoptosis- deficient BH3 domain mutant of Noxa, will be generated after determining that WT hNOXA promoter elements are functional and express hNoxa. If successfully generated, these mice could help validate hNoxa?s role in T cell metabolism during an immune response, offer insights into hNoxa?s role in leukemogenesis, and serve as pre-clinical models for testing therapeutic strategies to activate its pro-apoptotic function.